PROJECT SUMMARY AMPK is a three subunit kinase that functions as central regulator of energy homeostasis in all eukaryotes. Energy stress activates AMPK to globally reprogram cellular metabolism by turning on ATP-generating pathways, such as glucose and fatty acid uptake and catabolism, and turning down energy-consuming pathways, such as the synthesis of glycogen, glucose, fatty acids, cholesterol, and proteins, as well as cell growth and proliferation. AMPK is thus an important therapeutic target for the treatment of diabetes, obesity, and cancer, conditions under which AMPK is frequently inactivated or dysregulated. AMP directly activates, and ATP inhibits, AMPK 100- to 1,000-fold by inducing an AMPK conformation that stimulates phosphorylation, and inhibits dephosphorylation, of its activation loop. In addition, AMP also directly allosterically activates, and ATP inhibits, AMPK up to 10- fold. We and others have determined the mechanism of direct activation by AMP. However, understanding the distinct mechanism of direct inhibition by ATP, and most importantly of regulating activation loop phosphorylation and the conformational change between active and inactive states requires a structure of ATP-bound, inactive AMPK, which has remained elusive. Current specific pharmacological AMPK activators are biased to the ?1 isoform of AMPK, yet only activation of the ?2 isoform in muscle lowers blood glucose levels, which is critical for the treatment of diabetes and other metabolic diseases. The barrier to understanding the molecular basis, and overcoming, isotype specificity is the recalcitrance of activator-bound ?2 AMPK to crystallization. In this application we present a strategy, validated by preliminary crystals and negative stain EM 3D reconstructions, to determine the structures of AMPK in the inactive, ATP-bound state, as well as of ?2 holo-AMPK in complex with activating and inhibiting synthetic ligands. Comparison with our crystal structures of AMPK in AMP-bound, active state will allow us to identify conformational changes associated with AMPK activation and inhibition. We will validate the analysis of our static crystal structures and determine the conformational landscape of different AMPK states by dynamic hydrogen/deuterium exchange mass spectrometry, double electron- electron resonance (DEER) EPR, and by mutational and functional analyses. Together, the results of the proposed studies will provide a detailed mechanism of regulation of AMPK kinase activity and a structural basis for the rational design of therapeutics that can selectively activate or inhibit this important regulator.